L-arginine is a free-form amino acid found in plant seeds or garlic. L-arginine has been widely used as an efficient additive in medicaments, food, or the like. L-arginine is useful as a drug for improving the hepatic function and brain function, and treating male sterility, and as an ingredient of multiple amino acid supplements. Also, L-arginine has been used as a food additive in fish cakes and health beverages, and has recently gained interest as a salt substitute for hypertension patients.
Conventional methods for L-arginine production by biological fermentation are based on the production of L-arginine directly from carbon and nitrogen sources. For example, L-arginine can be produced using a mutant strain derived from a glutamic acid-producing microorganism belonging to the genus Brevibacterium or Corynebacterium (Japanese Unexamined Patent Publication Nos. Sho57-163487, Sho60-83593 and Sho62-265988), or using an amino acid-producing microorganism, of which growth properties are improved through cell fusion (Japanese Unexamined Patent Publication No. Sho59-158185). Recently, it has been reported that L-arginine can be produced using a recombinant strain, of which an argR gene that participates in regulation of arginine biosynthesis is inactivated (US Patent Application No. 2002/0045223A1) and using a method for over-expressing an argF gene of arginine operon (Korean Patent Application No. 10-2004-107215).
In microorganisms, biosynthesis of L-arginine proceeds in eight enzymatic steps starting from the precursor L-glutamate and follows two different pathways, the linear pathway or the cyclic pathway.
In microorganisms belonging to the genus Corynebacterium, L-arginine is synthesized through the cyclic pathway from L-glutamate via N-acetylglutamate, N-acetylglutamyl phosphate, N-acetylglutamate semialdehyde, N-acetylornithine, ornithine, citrulline and argininosuccinate. These intermediates are synthesized through consecutive reactions catalyzed by enzymes such as glutamate N-acetyltransferase, N-acetylglutamate kinase, acetylglutamate semialdehyde dehydrogenase, acetylornithine aminotransferase, ornithine cabomoyltransferase, arginosuccinate synthase, and arginosuccinate lyase. These enzymes are encoded by argJ, argB, argC, argD, argF, argG and argH genes, respectively.
In order to produce L-arginine in a high yield, the present inventors have conducted studies on the enzymes involved in L-arginine biosynthesis for a long period time. They found that in the intermediate step of arginine biosynthesis, the enzymatic reaction involved in the conversion of N-acetylglutamate semialdehyde to N-acetylornithine is amplified to increase L-arginine flux, thereby improving the productivity of L-arginine.
A variety of aminotransferases are present in cells, and divided into four subgroups on the basis of their mutual structural relatedness. Subgroup I comprises aspartylornithine, alanine, tyrosine, histidiolphosphate, and phenylalanine aminotransferases, subgroup II comprises acetylornithine, ornithine, ω-amino acid, aminobutyrate, and phenylalanine aminotransferases, subgroup III comprises D-alanine and branched-chain amino acid aminotransferases, and subgroup IV comprises serine and phosphoserine aminotransferases (Perdeep K. MEHTA, et al, Eur. J. Biochem., 214, 549-561, 1993).
It has been known that in Corynebacterium glutamicum, an argCJBDF gene involved in arginine biosynthesis exists as an operon, and is regulated by feedback-inhibition due to arginine (Vehary Sakanyan, et al, Microbiology, 142:9-108, 1996). Thus, there is a limit in producing L-arginine in a high yield.